A highly efficient identification of mutants generated by CRISPR/Cas9 using the non-functional DsRed assisted selection in Aspergillus oryzae

文献类型： 外文期刊

第一作者： Li, Yuzhen

作者： Li, Yuzhen;Fan, Junxia;Chen, Ziming;Chen, Tianming;Zeng, Bin;Zhang, Zhe;Li, Yuzhen;Fan, Junxia;Chen, Ziming;Chen, Tianming;Zeng, Bin;Zhang, Zhe;Zhang, Huanxin;Zeng, Bin

作者机构：

关键词： CRISPR; Cas9; Identification mutant; DsRed; Aspergillus oryzae

期刊名称：WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY （ 影响因子：3.312； 五年影响因子：3.58 ）

ISSN： 0959-3993

年卷期： 2021 年 37 卷 8 期

页码：

收录情况： SCI

摘要： The CRISPR/Cas9 system has become a great tool for target gene knock-out in filamentous fungi. It is laborious and time-consuming that identification mutants from a large number of transformants through PCR or enzyme-cut method. Here, we first developed a CRISPR/Cas9 system in Aspergillus oryzae using AMA1-based autonomously replicating plasmid and Cas9 under the control of the Aspergillus nidulans gpdA promoter. By the genome editing technique, we successfully obtained mutations within each target gene in Aspergillus oryzae. Then, we put the protospacer sequence of a target gene and its protospacer adjacent motif (PAM) behind the start codon "ATG" of DsRed, yielding the non-functional DsRed (nDsRed) reporter gene, and the nDsRed reporter gene could be rescued after successful targeted editing. Moreover, this method was also applied by targeting the kojic acid synthesis gene kojA, and the transformants with DsRed activity were found to harbor targeted mutations in kojA. These results suggest that the nDsRed can be used as a powerful tool to facilitate the identification of mutants generated by CRISPR/Cas9 in Aspergillus oryzae.

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